Investigation of corneal autoantibodies in horses with immune mediated keratitis (IMMK).

Immune mediated keratitis (IMMK) is primarily a non-ulcerative keratitis in horses causing intermittent ocular pain, eventually resulting in visual impairment. Affected horses typically respond to immunomodulatory treatment. However, the underlying cause of the disease remains enigmatic. The current study was undertaken to investigate the presence of autoantibodies in horses with immune mediated keratitis. Using 28 horses with IMMK and 27 healthy controls screening for serum autoantibodies against the corneal proteome using indirect immunofluorescence, one-dimensional (1DE) and two-dimensional electrophoresis (2DE) with subsequent western blot analysis was performed followed by mass spectrometric identification of bands or spots of interest. Indirect immunofluorescence did not reveal a difference in immune response towards corneal proteins between healthy horses and those with IMMK. Using western blot analysis some horses affected by IMMK (4/28) showed a single band (1D) or a single spot (2DE) (5/28) not detected in healthy controls. The corresponding spot was identified as maspin (SERPINB5), a protein responsible for the inhibition of corneal vascularisation, cell migration and cell adhesion to the extracellular matrix. Tests with a recombinant human protein commercially available did not verify blot findings, but the human protein may not be fully cross-reactive. Still, maspin might play a role in some cases of equine IMMK. Further research is needed to clarify the etiology of this disease.

In situ cell surface proteomics reveals differentially expressed membrane proteins in retinal pigment epithelial cells during autoimmune uveitis.

Retinal pigment epithelium (RPE) builds the outer blood-retinal barrier of the eye and plays an important role in pathogenesis of the sight threatening disease equine recurrent uveitis (ERU). ERU is a spontaneous autoimmune mediated inflammatory disease characterised by the breakdown of the outer blood-retinal barrier and an influx of autoaggressive T-cells into the inner eye. Therefore, identification of molecular mechanisms contributing to changed function of blood-retinal barrier in ERU is important for the understanding of pathophysiology. Cell surface proteins of RPE collected from healthy horses and horses with ERU were captured by in situ biotinylation and analysed with high resolution mass spectrometry coupled to liquid chromatography (LC-MS/MS) to identify differentially expressed proteins. With label free differential proteomics, a total of 27 differently expressed cell surface proteins in diseased RPE could be detected. Significant down-regulation of three very interesting proteins, synaptotagmin 1, basigin and collectrin was verified and further characterised. BIOLOGICAL SIGNIFICANCE: We applied an innovative and successful method to detect changes in the plasma cell surface proteome of RPE cells in a spontaneous inflammatory eye disease, serving as a valuable model for human autoimmune uveitis. We were able to identify 27 differentially expressed plasma cell membrane proteins, including synaptotagmin 1, basigin and collectrin, which play important roles in cell adhesion, transport and cell communication.

True blue: S-opsin is widely expressed in different animal species.

Colour vision in animals is an interesting, fascinating subject. In this study, we examined a wide variety of species for expression of S-opsin (blue sensitive) and M-/L-opsin (green-red sensitive) in retinal cones using two novel monoclonal antibodies specific for peptides from human opsins. Mouse, rat and hare did not express one of the investigated epitopes, but we could clearly prove existence of cones through peanut agglutinin labelling. Retinas of guinea pig, dog, wolf, marten, cat, roe deer, pig and horse were positive for S-opsin, but not for M-/L-opsin. Nevertheless all these species are clearly at least dichromats, because we could detect further S-opsin negative cones by labelling with cone arrestin specific antibody. In contrast, pheasant and char had M-/L-opsin positive cones, but no S-opsin expressing cones. Sheep, cattle, monkey, men, pigeon, duck and chicken were positive for both opsins. Visual acuity analyzed through density of retinal ganglion cells revealed least visual discrimination by horses and highest resolution in pheasant and pigeon. Most mammals studied are dichromats with visual perception similar to red-green blind people.

Follow-up protein profiles in urine samples during the course of obstructive feline idiopathic cystitis.

Feline idiopathic cystitis (FIC) is a common lower urinary tract disorder in cats, which often recurs. Published reports document increased urine fibronectin and thioredoxin concentrations in cats with FIC compared with healthy control cats. Therefore, these proteins might be of interest in the pathophysiology of FIC. The purpose of the present study was to evaluate variations in these urine proteins throughout the course of FIC by assessing their concentrations in urine specimens from cats with a history of obstructive FIC. Urine total protein (TP) was measured using the Bradford assay, while urine fibronectin and thioredoxin concentrations were determined by Western blot analysis. Urine TP was significantly higher in cats with obstructive FIC at presentation (day 0) than in healthy control cats (P<0.01). There were significant decreases in urine TP in cats with obstructive FIC after 3 months (P<0.01). Significantly higher urine fibronectin (P<0.01) and thioredoxin (P<0.05) concentrations were demonstrated in cats with FIC at day 0 compared to control cats, but there was no significant change over time (P>0.05). Increased concentrations of these proteins over time might reflect ongoing structural and pathological alterations to functional processes in the urinary bladders of cats with obstructive FIC.

Isolation, characterization and establishment of an equine retinal glial cell line: a prerequisite to investigate the physiological function of Müller cells in the retina.

Retinal Müller glial cells are of vital importance for maintaining a physiological environment within the retina. To this end, they provide highly specialized physiological properties to support neurons in structure, nutrition and metabolism. The purpose of this study was to isolate Müller cells from the equine retina, determine their characteristics and subsequently establish a stable equine Müller cell line (eqMC) that will provide a prerequisite for investigations on their physiological properties. Dissociated retinal cells were obtained from equine retinas by a papain digestion technique followed by trituration and a cell attachment method by which pure Müller cell cultures were achieved. Morphological examination was performed using phase-contrast microscopy, and further characterization of different subcultures was accomplished by immunocytochemistry. Cells of passage 1 showed distinct signals for glutamine synthetase and vimentin, whereas glial fibrillary acidic protein expression was almost absent. Characteristic expression patterns remained unaltered in all subcultures. Furthermore, cultured Müller cells stably expressed the microfilament alpha-smooth muscle actin, the proliferation marker Ki67 and the membrane channels Kir4.1 and aquaporin 4. The present study introduces the eqMC-7 that will facilitate studies investigating the physiological role of Müller cells within the equine retina.

[Blind spot enlargement syndrome in acute zonal occult outer retinopathy with detection of autoantibodies against the retinal antigens CRALBP and S-Ag]. / Syndrom der Vergrößerung des blinden Flecks in akuter zonaler okkulter äußerer Retinopathie mit Nachweis von Autoantikörpern gegen die retinalen Antigene CRALBP und S-Ag.

Acute zonal occult outer retinopathy (AZOOR) is a rare disease and is part of the white dot syndrome occurring bilaterally and often asymmetrically in young healthy myopic women. Characteristic findings are distinct focal lesions of the outer segments (OS) of the photoreceptor (PR) layer and abnormalities in fundus autofluorescence (FAF) within the lesions. Currently there is a lack of defined disease criteria, such as specific laboratory findings. Also no effective therapy is known which makes it difficult to diagnose, differentiate and treat AZOOR. Supplementation of antioxidants may become part of therapeutic options in AZOOR. A 19-year-old myopic woman presented with unilaterally reduced visual acuity. Due to the clinical features and with the help of FAF, spectral domain optical coherence tomography (SD-OCT) and perimetry the diagnosis of blind spot enlargement syndrome in AZOOR was made. Identification of autoantibodies specific for two retinal antigens (CRALBP and S-Ag) supports the concept of an autoimmunological origin of the disease. Systemic steroids were given but stopped almost 6 weeks later as no improvement was seen. In follow-up controls over 12 months the clinical picture remained unchanged without any further therapy.

Defining cytochemical markers for different cell types in the equine retina.

The major cell types in the mammalian retina are photoreceptors, amacrine, horizontal, bipolar, ganglion and Mueller glial cells. Most of the specific cell types are conserved, but cytochemical markers vary between species. The aim of our study was to characterize cytochemically distinctive markers for different cell types in the equine retina. We were able to define specific markers for equine Mueller glial cells and photoreceptor cells. Furthermore, we describe markers for large ganglion cells, horizontal and amacrine cells and a subpopulation of bipolar cells. Additionally, discrimination between the inner plexiform layer and nerve fibre layer can be achieved by expression of syntaxin and neurofilament 200 respectively.

Immunopathology of recurrent uveitis in spontaneously diseased horses.

Equine recurrent uveitis (ERU) is the most serious eye disease in horses worldwide. Despite the fact that ERU is generally considered to be immune mediated, a detailed description of the histopathology of the posterior part of ERU eyes is lacking. Here, we examined sections of paraffin-embedded eyes using histological and immunhistological methods. Twenty seven eyes of 20 horses with ERU and 30 eyes of 15 healthy control horses were included in this study. We could consistently demonstrate an involvement of the retina and the choroid in all examined eyes of horses with spontaneous ERU. In eyes with minimal histopathological changes, the infiltrates consisted almost exclusively of T-cells. Histopathological changes start with the destruction of the photoreceptor outer segments, which often leads to focal retinal detachment. In more severely affected eyes, there is additional disintegration of the ganglion cell layer and the inner nuclear layer. In almost all examined eyes, lymphoid follicle formation could be demonstrated. Typical localizations of these follicles were the iris stroma and the choroid underneath the transition zone of the retina without photoreceptor cells to the region containing photoreceptor cells. These follicles consist of a T-cell rich periphery with a small center of CD3-negative lymphocytes. In cases with extreme histopathological changes, the retinal architecture is widely disintegrated with massive infiltration of the retina, the choroid, and the ciliary body by several types of inflammatory cells. Necrotic remnants of the retina are end-stage findings and there is only a minor inflammatory infiltration left. This study provides clear evidence that the retina is involved in all stages of ERU. Inflammation is mainly driven by T-cells as T-cells were demonstrated in mild stages of the disease and are also the predominating cell type in all other stages of ERU.

Immune responses to retinal autoantigens and peptides in equine recurrent uveitis.

PURPOSE: To test the hypothesis that autoimmune mechanisms are involved in horses in which equine recurrent uveitis (ERU) develops spontaneously. METHODS: Material obtained from horses treated for spontaneous disease by therapeutic routine vitrectomy was analyzed for total IgG content and IgG specific for S-Antigen (S-Ag) and interphotoreceptor retinoid-binding protein (IRBP). The cellular infiltrate of the vitreous was analyzed by differential counts of cytospin preparations and flow cytometry using equine lymphocyte-specific antibodies. Antigen-specific proliferation assays were performed comparing peripheral blood lymphocytes (PBLs) with vitreal lymphocytes by stimulation with S-Ag and several S-Ag- and IRBP-derived peptides. RESULTS: The total IgG content of specimens from horses with ERU was very high with great variability among the investigated samples (11.5 +/- 8.0 mg). Autoantibodies to S-Ag or IRBP or both were found in 72% of vitreous specimens from horses with uveitis. The leukocyte infiltrates (up to 2 x 10(8) cells per sample) were dominated by lymphocytes (>90%) in most cases (22/32). Flow cytometry showed that more than 50% of these cells were CD4(+) T cells. In vitro stimulation of vitreal lymphocytes, but not of PBL, showed a strong proliferative response to peptides derived from S-Ag or IRBP in 9 of 12 patients. CONCLUSIONS: In the eyes of horses with ERU, IgG antibodies and autoreactive T cells specific for retinal antigens were detected. These results strongly support the hypothesis that ERU is an autoimmune-mediated disease and is highly similar to recurrent uveitis in humans in both clinical and immunologic parameters.